Redefining the Minimal Substrate Tolerance of Mandelate Racemase. Racemization of Trifluorolactate

Abstract

Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the enantiomers of mandelic acid and a variety of aryl- and heteroaryl-substituted mandelate derivatives, suggesting that β,γ-unsaturation is a requisite feature of substrates for the enzyme. We show that β,γ-unsaturation is not an absolute requirement for catalysis and that mandelate racemase can bind and catalyze the racemization of (S)-trifluorolactate (k(cat) = 2.5 ± 0.3 s(-1), K(m) = 1.74 ± 0.08 mM) and (R)-trifluorolactate (k(cat) = 2.0 ± 0.2 s(-1), K(m) = 1.2 ± 0.2 mM). The enzyme was shown to catalyze hydrogen-deuterium exchange at the α-postion of trifluorolactate using (1)H NMR spectrocsopy. β-Elimination of fluoride was not detected using (19)F NMR spectroscopy. Although mandelate racemase bound trifluorolactate with an affinity similar to that exhibited for mandelate, the turnover numbers (k(cat)) were markedly reduced by ∼318-fold, resulting in catalytic efficiencies (k(cat)/K(m)) that were ~400-fold lower than those observed for mandelate. These observations suggested that chemical steps on the enzyme were likely rate-determining, which was confirmed by demonstrating that the rates of mandelate racemase-catalyzed racemization of (S)-trifluorolactate were not dependent upon the solvent microviscosity. Circular dichroism spectroscopy was used to measure the rates of nonenzymatic racemization of (S)-trifluorolactate at elevated temperatures. The values of ΔH(‡) and ΔS(‡) for the nonenzymatic racemization reaction were determined to be 28.0 (±0.7) kcal/mol and -15.7 (±1.7) cal K(-1) mol(-1), respectively, corresponding to a free energy of activation equal to 33 (±4) kcal/mol at 25 °C. Hence, mandelate racemase stabilizes the altered trifluorolactate in the transition state (ΔG(tx)) by at least 20 kcal/mol.

Lactate racemase is a nickel-dependent enzyme activated by a widespread maturation system

“…Besides lactate racemase, the only known α-hydroxyacid racemase is the mandelate racemase, which is a Mg-dependent enzyme of the enolase superfamily 11 . The majority of racemases are amino acid racemases, which are either pyridoxal 5′-phosphate (PLP) dependent or PLP-independent enzymes 12 .…”

“…The majority of racemases are amino acid racemases, which are either pyridoxal 5′-phosphate (PLP) dependent or PLP-independent enzymes 12 . Both mandelate racemase and PLP-independent racemases rely on intramolecular stabilization of a deprotonated reaction intermediate for their catalysis 11 , 13 Concerning the lactate racemase, another mechanism is probably taking place due to the absence of an electron-withdrawing group on lactate. A few reports have addressed the Lar activity of Lactobacillus sakei , C. beijerinckii and C. acetobutylicum and suggested a hydride transfer mechanism 14 - 17 .…”

Racemases and epimerases operating through a 1,1-proton transfer mechanism: reactivity, mechanism and inhibition

“…Exceptions to this strategy are seen with methylmalonyl-CoA epimerase 90 and mandelate racemase, 91,92 where the carboxylate group is ligated to the active site Co 2+ or Mg 2+ ion which acts as a Lewis acid and diminishes the pK a of the C a -H. 21 Typically the carboxylate group is also held within a hydrogen-bonding network with active-site residues. 5 Some racemase/epimerase substrates also contain further destabilising groups, such as ammonium groups (amino-acid racemases/epimerases), 18,20 amide carbonyl groups (N-succinylamino acid racemases, dipeptide epimerases and other enolase family enzymes 18,49 ) and OH (mandelate racemase, 18,91 various sugar epimerases 18 ). Both ammonium and OH groups are more easily deprotonated than the C a -H. Chemical models 86,93 show that the pK a of the C a -H is diminished by 9-15 units by protonation of an adjacent amine and a number of amino-acid racemases/epimerases, 20,94 including diaminopimelate epimerase and glutamate racemase, appear to protonate the amine of the substrate during the reaction.…”

“…Both ammonium and OH groups are more easily deprotonated than the C a -H. Chemical models 86,93 show that the pK a of the C a -H is diminished by 9-15 units by protonation of an adjacent amine and a number of amino-acid racemases/epimerases, 20,94 including diaminopimelate epimerase and glutamate racemase, appear to protonate the amine of the substrate during the reaction. In the case of mandelate racemase 18,91 and N-succinylaminoacid racemases, 49 the OH or amide carbonyl groups are ligated to active-site metals such as Mg 2+ (mandelate racemase 18,91,92 ) or Co 2+ , Mn 2+ or, occasionally, Mg 2+ (N-succinylamino-acid racemases 49 ). The rates of proton transfer for the deprotonation and reprotonation steps are generally high, with rate constants of the order of 5 Â 10 9 to 100 Â 10 9 M À1 s À1 .…”

“…b-Elimination to give 22 is not observed (Scheme 4B). 91 L. aggregata cis-3-hydroxy-S-proline racemase/dehydratase (IAM 12614) 119 catalyses both racemisation and b-elimination reactions with its substrate 23, in a 3 to 2 ratio (Scheme 5). The b-elimination reaction is proposed to go via an enediolate intermediate 24, although it may be a more concerted E2-like reaction.…”